The overall goal of this proposal is to study the involvement of the peptide substance P (SP) in the pathogenesis of enterotoxin-mediated diarrhea, inflammation and mucosal damage. Our recent studies with a specific SP antagonist have provided evidence for an important role of SP, a major constituent of primary sensory neurons, in these pathological processes. Immune cells in the lamina propria contain SP and express mRNAs for SP encoding both SP and its receptor (SPR). Immune cells could thus be another source of SP and function in a paracrine and/or autocrine manner in regulating gastrointestinal inflammatory processes. A recent exciting observation is that intestinal epithelial cells express SPR mRNA, the content of SPR mRNA is dramatically increased by exposure to toxin A and this increase is inhibited by pretreament of rats with the SP antagonist 96,345. Thus, these cells may participate in the inflammatory response to toxin A. The role of SP in these processes in the intestine will be assessed in a rat ileal loop model using Clostridium difficile enterotoxin (toxin A)-induced diarrhea and inflammation. In aim 1 we will study cellular localization of SP and SP receptor (SPR) and expression of SPmRNA and SPRmRNA in epithelial cells and immune cells of the lamina propria before and after exposure to toxin A. We will also determine whether the immune cells that invade the intestine after toxin A administration also contain SP, SP mRNA and SPR mRNA. In aim 2 we will examine the effect of SP on cytokine release (interleukin-l, and tumor necrosis factor alpha) from purified lamina propria macrophages (LPMs) and from intestinal tissue exposed to toxin A. In aim 3 we will evaluate the role of SP in activation of neutrophils in toxin A mediated enteritis. The binding of fluorescein- labelled SP to PMNs also will be characterized. In aim 4 we will examine a role for SP in intestinal mast cell responses after toxin A-induced secretion and release of mediators (histamine, prostaglandin D2, leukotriene C4 and rat mast cell protease II). The inflammatory effects of toxin A will also be studied in a strain of mice (WBB6F1-W/Wv) which are mast cell deficient. Results from these studies will help delineate the mechanisms of SP-mediated intestinal secretion and inflammation and provide information on a new important therapeutic approach to intestinal inflammatory diseases.